The erythropoietin receptor (EpoR) is the primary regulator of mammalian erythropoiesis. The EpoR lacks intrinsic catalytic activity and relies on Janus Kinase 2 (JAK2) for signal transduction. Homo-dimerization of the EpoR in response to erythropoietin activates JAK2 kinase activity and in turn triggers downstream signal transduction. Aberrant activation of the EpoR can be oncogenic and lead to leukemia. Several lines of evidence show that JAK2 is an essential subunit of the EpoR and should be considered together with the EpoR as a functional entity. Unfortunately, little information is known about the structures of the cytoplasmic domain of any cytokine receptors, JAKs, or their complexes. This fellowship will probe the molecular structure of the EpoR/JAK2 complex. To accomplish this, biochemical, biophysical, and functional assays will be used. A novel functional assay will be used to study the functional interactions between the EpoR and JAK2. Deletion mutations on both the EpoR and on JAK2, and chimeric proteins between JAK1 and JAK2 will help map the minimal segments on the EpoR and on JAK2 for their interaction. Protein footprinting experiments will identify global interaction surfaces and potential conformational changes in the EpoR/JAK2 complex. Four specific residues in the cytoplasmic domain of the EpoR have been identified not for binding JAK2 but for its activation. These residues may interact with JAK2 specifically to switch on the kinase activity. These key residues will be mutated to cysteines on a cysteine deficient mutant EpoR and examined for protection against chemical modification upon binding JAK2 or domains of JAK2 using thio-specific reagents. Comparison between the accessibilities with or without JAK2 or domains of JAK2 will identify regions on JAK2 that interact with these specific amino acids. These results will help delineate the functional roles of specific key residues and also help construction of a three dimensional model for the EpoR/JAK2 complex. Understanding of the molecular structure of the EpoR/JAK2 complex will lead to a deeper and more comprehensive understanding of how EpoR-mediated signal transduction regulates the differentiation and proliferation of hematopoietic cells. This information will help to understand how an oncogenic EpoR induces leukemia in mice, and will shed considerable light on intracellular signal transduction pathways by other cytokines. Through this research proposal, the applicant will receive training in biophysical and structural biology and will gain the necessary skills and knowledge to become an independent investigator.